Background:The role of the circadian protein Per1 in the regulation of sodium reabsorption in the distal convoluted tubule (DCT) is unknown. Results: Per1 transcriptionally regulates the sodium transporter NCC and the WNK kinase cascade. Conclusion: Per1 regulates sodium reabsorption in the DCT through NCC and the WNK cascade.Significance: These data demonstrate a role for Per1 in the regulation of renal sodium transporters.
The circadian clock plays an important role in the regulation of physiological processes, including renal function and blood pressure. We have previously shown that the circadian protein period (Per)1 regulates the expression of multiple Na+ transport genes in the collecting duct, including the α-subunit of the renal epithelial Na+ channel. Consistent with this finding, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. We have also recently demonstrated the potential opposing actions of cryptochrome (Cry)2 on Per1 target genes. Recent work by others has demonstrated that Cry1/2 regulates aldosterone production through increased expression of the adrenal gland-specific rate-limiting enzyme 3β-dehydrogenase isomerase (3β-HSD). Therefore, we tested the hypothesis that Per1 plays a role in the regulation of aldosterone levels and renal Na+ retention. Using RNA silencing and pharmacological blockade of Per1 nuclear entry in the NCI-H295R human adrenal cell line, we showed that Per1 regulates 3β-HSD expression in vitro. These results were confirmed in vivo: mice with reduced levels of Per1 had decreased levels of plasma aldosterone and decreased mRNA expression of 3β-HSD. We postulated that mice with reduced Per1 would have a renal Na+-retaining defect. Indeed, metabolic cage experiments demonstrated that Per1 heterozygotes excreted more urinary Na+ compared with wild-type mice. Taken together, these data support the hypothesis that Per1 regulates aldosterone levels and that Per1 plays an integral role in the regulation of Na+ retention.
Mounting evidence suggests that the circadian clock plays an integral role in the regulation of many physiological processes including blood pressure, renal function, and metabolism. The canonical molecular clock functions via activation of circadian target genes by Clock/Bmal1 and repression of Clock/Bmal1 activity by Per1–3 and Cry1/2. However, we have previously shown that Per1 activates genes important for renal sodium reabsorption, which contradicts the canonical role of Per1 as a repressor. Moreover, Per1 knockout (KO) mice exhibit a lowered blood pressure and heavier body weight phenotype similar to Clock KO mice, and opposite that of Cry1/2 KO mice. Recent work has highlighted the potential role of Per1 in repression of Cry2. Therefore, we postulated that Per1 potentially activates target genes through a Cry2-Clock/Bmal1-dependent mechanism, in which Per1 antagonizes Cry2, preventing its repression of Clock/Bmal1. This hypothesis was tested in vitro and in vivo. The Per1 target genes αENaC and Fxyd5 were identified as Clock targets in mpkCCDc14 cells, a model of the renal cortical collecting duct. We identified PPARα and DEC1 as novel Per1 targets in the mouse hepatocyte cell line, AML12, and in the liver in vivo. Per1 knockdown resulted in upregulation of Cry2 in vitro, and this result was confirmed in vivo in mice with reduced expression of Per1. Importantly, siRNA-mediated knockdown of Cry2 and Per1 demonstrated opposing actions for Cry2 and Per1 on Per1 target genes, supporting the potential Cry2-Clock/Bmal1-dependent mechanism underlying Per1 action in the liver and kidney.
We have previously demonstrated that the circadian clock protein period (Per)1 coordinately regulates multiple genes involved in Na(+) reabsorption in renal collecting duct cells. Consistent with these results, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. The proximal tubule is responsible for a majority of Na(+) reabsorption. Previous work has demonstrated that expression of Na(+)/H(+) exchanger 3 (NHE3) oscillates with a circadian pattern and Na(+)-glucose cotransporter (SGLT)1 has been demonstrated to be a circadian target in the colon, but whether these target genes are regulated by Per1 has not been investigated in the kidney. The goal of the present study was to determine if Per1 regulates the expression of NHE3, SGLT1, and SGLT2 in the kidney. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of SGLT1 and NHE3 but not SGLT2 in the renal cortex of mice. Per1 small interfering RNA and pharmacological blockade of Per1 nuclear entry in human proximal tubule HK-2 cells yielded the same results. Examination of heterogeneous nuclear RNA suggested that the effects of Per1 on NHE3 and SGLT1 expression occurred at the level of transcription. Per1 and the circadian protein CLOCK were detected at promoters of NHE3 and SGLT1. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased after blockade of nuclear Per1 entry. This effect was associated with reduced activity of Na(+)-K(+)-ATPase. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney.
Aims The present study is designed to consider a role for the circadian clock protein Per1 in the regulation of the endothelin axis in mouse kidney, lung, liver and heart. Renal endothelin-1 (ET-1) is a regulator of the epithelial sodium channel (ENaC) and blood pressure (BP), via activation of both endothelin receptors, ETA and ETB. However, ET-1 mediates many complex events in other tissues. Main methods Tissues were collected in the middle of murine rest and active phases, at noon and midnight, respectively. ET-1, ETA and ETB mRNA expressions were measured in the lung, heart, liver, renal inner medulla and renal cortex of wild type and Per1 heterozygous mice using real-time quantitative RT-PCR. Key findings The effect of reduced Per1 expression on levels of mRNAs and the time-dependent regulation of expression of the endothelin axis genes appeared to be tissue-specific. In the renal inner medulla and the liver, ETA and ETB exhibited peaks of expression in opposite circadian phases. In contrast, expressions of ET-1, ETA and ETB in the lung did not appear to vary with time, but ET-1 expression was dramatically decreased in this tissue in Per1 heterozygous mice. Interestingly, ET-1 and ETA, but not ETB, were expressed in a time-dependent manner in the heart. Significance Per1 appears to regulate expression of the endothelin axis genes in a tissue-specific and time-dependent manner. These observations have important implications for our understanding of the best time of day to deliver endothelin receptor antagonists.
Renal function and blood pressure (BP) exhibit a circadian pattern of variation, but the molecular mechanism underlying this circadian regulation is not fully understood. We have previously shown that the circadian clock protein Per1 positively regulates the basal and aldosterone-mediated expression of the alpha subunit of the renal epithelial sodium channel (αENaC). The mechanism of this regulation has not been determined however. To further elucidate the mechanism of mineralocorticoid receptor (MR) and Per1 action, site-directed mutagenesis, DNA pull-down assays and chromatin immunoprecipitation (ChIP) methods were used to investigate the coordinate regulation of αENaC by Per1 and MR. Mutation of two circadian response E-boxes in the human αENaC promoter abolished both basal and aldosterone-mediated promoter activity. DNA pull down assays demonstrated the interaction of both MR and Per1 with the E-boxes from the αENaC promoter. These observations were corroborated by ChIP experiments showing increased occupancy of MR and Per1 on an E-box of the αENaC promoter in the presence of aldosterone. This is the first report of an aldosterone-mediated increase in Per1 on a target gene promoter. Taken together, these results demonstrate the novel finding that Per1 and MR mediate the aldosterone response of αENaC through DNA/protein interaction in renal collecting duct cells.
VMAT CSI, along with chemotherapy and anesthesia, is feasible with supportive care. Daily image-guided RT improves accuracy and reduces the risk of spinal cord overdose without increasing treatment time. Further research is needed to determine whether reducing doses to organs, such as thyroid, heart, or hippocampus, offsets the risk of increased volume of low-dose irradiation.
The purpose of this work was to compare dose distributions between two radiosurgery modalities, single‐isocenter volumetric modulated arc therapy (VMAT), and GammaKnife Perfexion (GK), in the treatment of a large number (≥7) of brain metastases. Twelve patients with 103 brain metastases were analyzed. The median number of targets per patient was 8 (range: 7–14). GK plans were compared to noncoplanar VMAT plans using both 6‐MV flattening filter‐free (FFF) and 10‐MV FFF modes. Parameters analyzed included radiation therapy oncology group conformity index (CI), 12, 6, and 3 Gy isodose volumes (V12 Gy, V6 Gy, V3 Gy), mean and maximum hippocampal dose, and maximum skin dose. There were statistically significant differences in CI (2.5 ± 1.6 vs 1.6 ± 0.8 and 1.7 ± 0.9, P < 0.001, P < 0.001), V12 Gy (2.8 ± 6.1 cc vs 3.0 ± 5.2 cc and 3.1 ± 5.4 cc, P = 0.003, P < 0.001), and V3 Gy (323.0 ± 294.8 cc vs, 880.1 ± 369.1 cc and 937.9 ± vs 361.9 cc, P = 0.005, P = 0.001) between GK versus both 6‐MV FFF and 10‐MV FFF. No significant differences existed for maximum hippocampal or skin doses. In conclusion, highly optimized VMAT produced improved conformity at the expense of a higher V12 Gy and V3 Gy volume when compared with highly optimized GK.
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